The present invention relates to magnetic metal(loid) oxide-containing spherical silica gel particles having high nucleic acid binding capacity, to a process for their preparation and to their use in the bioanalytical and diagnostic sector.
Silica particles have been used for years in bioanalysis for removing and purifying nucleic acids, being particularly able because of their specific physicochemical structure to bind these nucleic acids. Such media which can be employed exclusively in column chromatography are described in German patent DE 32 11 309 (corresponding to U.S. Pat. No. 4,699,717).
The PCT application EP99/08996 describes glass-coated pigments for nucleic acid purification which comprise various metal oxides such as zinc, boron, iron, calcium, potassium and/or aluminum. Glass particles having a mica core and incorporated magnetite particles which are, however, prone to rapid sedimentation are revealed in the PCT application EP96/02459. The preparation processes are time-consuming and require technically elaborate spray drying processes. Ideally spherical particles cannot be prepared by these processes.
Anal. Biochem. 201, 166 (1992) and PCT GB91/00212 describe nucleic acid separation processes using magnetic particles which are able to absorb the nucleic acids after a salt-ethanol precipitation. However, these processes do not operate nucleic acid-specifically, i.e. the magnetic particles also absorb other biosubstances in parallel.
Silanized iron oxide particles for immobilizing enzymes are disclosed in U.S. Pat. No. 4,152,210. Ferromagnetic particles likewise for the purpose of enzyme immobilization are described in U.S. Pat. No. 4,343,901 and are prepared by a sol-gel technique.
The PCT application EP97/04828 describes monodisperse magnetic particles which consist of an SiO2 core which acquires magnetic properties by coating with iron oxide. The particles are enabled to bind nucleic acids by subsequent silanization of the iron oxide layer. U.S. Pat. No. 5,320,944 discloses, analogously thereto, magnetic particles which are 0.2-3 μm in size and which acquire magnetic properties by coating a polymer particle with iron oxides. Further coating of the particles with silanes, nylon or polystyrene subsequently allows antibodies to be coupled to the particles for use in immunoassays. Iron oxide particles coated with colloidal SiO2 are disclosed in U.S. Pat. No. 4,280,918.
Magnetic silica hybrid particles consisting of a polystyrene core onto which magnetite and subsequently a silicon layer are polymerized are disclosed in PCT/US 95/12988. The particles are employed for separating antibodies and cells.
Magnetic silica gel particles 20-100 μm in size for enzyme immobilization, which are generated by electrostatic coating of nickel powders with silica sols, have been described by Goetz et al., Biotechn. & Bioengineering, Vol. 37, 614, 1991.
Organosilanized colloidal silica gel particles as biological separation media are disclosed in the PCT application US99/00403, where the stability of the colloids and the mode of silanization have priority. Magnetic particles which comprise a magnetic core material and are coated with an inorganic oxide are disclosed in EP 0 343 934.
Polymer particles coated with a polymer layer comprising magnetic substances, to which a third polymer coating able to interact with biomolecules is applied are described in PCT application FR97/00912.
Pearl luster color pigments which are 10-60 μm in size and which are enveloped with magnetite and are intended for separating biological mixtures are revealed in the PCT application DE97/01300 (corresponding to U.S. Pat. No. 6,372,517).
U.S. Pat. No. 5,648,124 relates to magnetic hybrid particles which consist of a polymer core and which are first coated with a ferrofluid and subsequently coated with a functional polyacrylate.
U.S. Pat. Nos. 6,204,033 and 6,514,688 describe spherical magnetic polyvinyl alcohol-based polymer particles which can be prepared by inverse suspension polymerization within a short time. The polymer particles disclosed therein are, however, unsuitable for nucleic acid purification without extensive derivatization steps because of the physicochemical properties of polyvinyl alcohol.
The particles known in the art have some disadvantages in relation to the removal of nucleic acids, if they are in fact suitable for this application: firstly, a number of support media are not magnetic (U.S. Pat. No. 4,927,750, DE 32 11 309, PCT/US99/00403; PCT/EP94/01378) so that rapid removal of the particles, as is now required in automated routine analyses, is impossible. Secondly, silica- or polystyrene-based magnetic particles which are coated with a magnetic oxide have a high specific density (PCT/EP97/04828, U.S. Pat. No. 4,152,210, EP 3211309, U.S. Pat. No. 5,320,944), resulting in inadequate dispersibility together with rapid settling of the particles. Use of these particles in an immunoassay or nucleic acid assay which is predominantly carried out in suspension is thus adversely affected because additional mechanical mixing is required. The crucial disadvantage of the coated particles is, however, that the metal oxides may, both as core material and as coating material, despite the subsequent silanization, come into direct contact with the analytical solution. This represents a serious problem in the analysis of nucleic acids, e.g. within the framework of PCR, because the polymerases used in PCR may be deactivated in contact with metals.
The processes known in the art for producing the magnetic particles are always very complicated and, without exception, require a production process lasting many hours.
In addition, the PCT application EP01/08392, corresponding to DE 100 35 953 A1, discloses an inverse suspension process for preparing silica particles which is able to avoid the disadvantages evident from the prior art in relation to the material circumstances and/or the expenditure of time and experimental effort. This process, which is incorporated herein by reference, is based on magnetic colloid-containing aqueous silica sols which are dispersed in specific organic phases and are consolidated to spherical gel particles by addition of base during the dispersion procedure. The disadvantage of the silica gels produced by this process is, however, that they are hydrogels which, as a result of the high water content, are very polar or hydrophilic, which adversely affects the nucleic acid binding capacity. In addition, no support modifications specifically assisting nucleic acid binding are described, so that use of the supports in purifying nucleic acids is unsatisfactory because of the low binding capacity.